Effect of frozen-thawed embryo transfer with a poor-quality embryo and a good-quality embryo on pregnancy and neonatal outcomes.

BACKGROUND: To evaluate the impact of embryo quality and quantity, specifically a poor quality embryo (PQE) in combination with a good quality embryo (GQE), by double embryo transfer (DET) on the live birth rate (LBR) and neonatal outcomes in patients undergoing frozen-thawed embryo transfer (FET) cycles. METHODS: A study on a cohort of women who underwent a total of 1462 frozen-thawed cleavage or blastocyst embryo transfer cycles with autologous oocytes was conducted between January 2018 and December 2021. To compare the outcomes between single embryo transfer (SET) with a GQE and DET with a GQE and a PQE, propensity score matching (PSM) was applied to control for potential confounders, and a generalized estimating equation (GEE) model was used to determine the association between the effect of an additional PQE and the outcomes. Subgroup analysis was also performed for patients stratified by female age. RESULTS: After PS matching, DET-GQE + PQE did not significantly alter the LBR (adjusted odds ratio [OR] 1.421, 95% CI 0.907-2.228) compared with SET-GQE in cleavage-stage embryo transfer but did increase the multiple birth rate (MBR, [OR] 3.917, 95% CI 1.189-12.911). However, in patients who underwent blastocyst-stage embryo transfer, adding a second PQE increased the live birth rate by 7.8% ([OR] 1.477, 95% CI 1.046-2.086) and the multiple birth rate by 19.6% ([OR] 28.355, 95% CI 3.926-204.790), and resulted in adverse neonatal outcomes. For patients who underwent cleavage-stage embryo transfer, transferring a PQE with a GQE led to a significant increase in the MBR ([OR] 4.724, 95% CI 1.121-19.913) in women under 35 years old but not in the LBR ([OR] 1.227, 95% CI 0.719-2.092). The increases in LBR and MBR for DET-GQE + PQE compared with SET-GQE in women older than 35 years were nonsignificant toward. For patients who underwent blastocyst-stage embryo transfer, DET-GQE + PQE had a greater LBR ([OR] 1.803, 95% CI 1.165-2.789), MBR ([OR] 24.185, 95% CI 3.285-178.062) and preterm birth rate (PBR, [OR] 4.092, 95% CI 1.153-14.518) than did SET-GQE in women under 35 years old, while no significant impact on the LBR ([OR] 1.053, 95% CI 0.589-1.884) or MBR (0% vs. 8.3%) was observed in women older than 35 years. CONCLUSIONS: The addition of a PQE has no significant benefit on the LBR but significantly increases the MBR in patients who underwent frozen-thawed cleavage-stage embryo transfer. However, for patients who underwent blastocyst-stage embryo transfer, DET-GQE + PQE resulted in an increase in both the LBR and MBR, which may lead to adverse neonatal outcomes. Thus, the benefits and risks of double blastocyst-stage embryo transfer should be balanced. In patients younger than 35 years, SET-GQE achieved satisfactory LBR either in cleavage-stage embryo transfer or blastocyst-stage embryo transfer, while DET-GQE + PQE resulted in a dramatically increased MBR. Considering the low LBR in women older than 35 years who underwent single cleavage-stage embryo transfer, selective single blastocyst-stage embryo transfer appears to be a more promising approach for reducing the risk of multiple live births and adverse neonatal outcomes.

Dexamethasone application for in vitro fertilisation in non-classic 17-hydroxylase/17,20-lyase-deficient women.

Context: High progesterone levels in the follicular stage interfere with the implantation window, causing infertility in women with 17-hydroxylase/17,20-lyase deficiency (17OHD). Dexamethasone can restore cortisol deficiency and suppress inappropriate mineralocorticoid secretion to control hypertension in 17OHD patients, but poses risks to the foetus if administered during pregnancy. Objective: We prospectively explored a rational glucocorticoid use protocol for assistive reproduction in a woman with non-classic 17OHD that reduced glucocorticoid side effects. Method: In this study, the treatment protocol for this 17OHD patient included the following steps. First, the appropriate type and dose of glucocorticoid for endogenous progesterone suppression was determined. Then, glucocorticoid was discontinued to increase endogenous progesterone levels for ovarian stimulation. Next, dexamethasone plus GnRHa were used to reduce progesterone levels in frozen embryos for transfer. Once pregnancy was confirmed, dexamethasone was discontinued until delivery. Results: Dexamethasone, but not hydrocortisone, reduced progesterone levels in the 17OHD woman. After endogenous progesterone-primed ovarian stimulation, 11 oocytes were retrieved. Seven oocytes were 2PN fertilised and four day-3 and two day-5 embryos were cryopreserved. After administering dexamethasone plus gonadotropin-releasing hormone agonist (GnRHa) to reduce progesterone levels to normal, hormone replacement therapy was administered until the endometrial width reached 9 mm. Exogenous progesterone (60 mg/day) was used for endometrial preparation. Two thawed embryos were transferred on day 4. Dexamethasone was continued until pregnancy confirmation on the 13th day post-transfer. Two healthy boys, weighing 2100 and 2000 g, were delivered at 36 weeks' gestation. Conclusion: Rational use of dexamethasone synchronised embryonic development with the endometrial implantation window, while not using in post-implantation avoided its side effects and promoted healthy live births in women non-classic 17OHD undergoing in vitro fertilisation.

The presence of ovarian endometrioma adversely affect ovarian reserve and response to stimulation but not oocyte quality or IVF/ICSI outcomes: a retrospective cohort study.

BACKGROUND: The possible impact of ovarian endometriomas (OMAs) on in vitro fertilization (IVF) outcomes remains controversial. Therefore, this study aimed to assess the impact of OMAs on IVF cycle parameters, including ovarian reserve and response to stimulation, embryo quality and pregnancy outcomes. METHODS: This retrospective cohort study included 2067 patients undergoing their first IVF/ICSI cycles between January 2018 and December 2020. The study group included 154 infertile women who had OMAs. The control group consisted of 1913 women without endometriosis, and finally 305 women were matched according to maternal age, body mass index (BMI), and infertility duration by propensity score matching (PSM). Cumulative live birth rate (CLBR) was set as the primary outcome measure. Logistic regression analysis was conducted on the basis of clinical covariates assessed for their association with CLBRs. Subgroup analyses were performed to evaluate the effect of ovarian surgery, cyst size and laterality on CLBRs. RESULTS: Women with OMAs had significantly lower ovarian reserve markers (AMH and AFC), number of follicles, oocytes, embryos, and top-quality embryos than women in the control group (p < 0.05). However, the CLBRs were comparable between the two groups (55.64% versus 54.34%, p = 0.806), regardless of previous history of ovarian surgery. Multivariate analysis revealed association between age (OR = 0.861; 95% CI [0.806-0.921]; p = 0.000), top-quality embryos (OR = 1.829; 95% CI [1.526-2.193]; p = 0.000) and the CLBRs. A negative correlation between OMA size and AFC levels in patients with unoperated OMAs was detected (r = -0.264, p = 0.007). Meanwhile, significant decrease in ovarian reserve with lower AFC, fewer oocytes, embryos and top-quality embryos were observed in patients with OMAs size ≥ 6 cm (p < 0.05). Moreover, ovaries with OMAs had a significantly lower AFC (P = 0.006) but similar number of oocytes when compared with contralateral ovaries without OMAs. CONCLUSION: Infertile women with OMAs were implicated in considerable decreases in ovarian reserve and response to stimulation, but no apparent adverse effects on oocyte quality or clinical outcomes. OMAs surgery and OMAs size may adversely affect ovarian reserve, but not CLBR.

The Inclusion Principles of Human Embryos in the WOW-Based Time-Lapse System: A Retrospective Cohort Study.

A time-lapse system (TLS) with a well-of-the-well (WOW) dish, which allows individual identification and the possibility of autocrine and paracrine signaling between group-cultured embryos, has been widely used in clinic. However, there is a need to re-think the inclusion principles of human embryos in WOW-based TLS, especially for grade IV (G4) embryos, which are considered to potentially have detrimental effects on surrounding embryos. Here, we carried out a single-center, large-cohort, retrospective study, comprising 303 patients undergoing IVF (148 cases) and ICSI (155 cases), with a total of 3282 embryos, to compare embryonic development until the blastocyst stage in the group culture system with or without G4 embryos. Further, LC-MS/MS was used to analyze the G1-G4 embryo secretome to understand the influence of G4 embryos on the group culture microenvironment. We proved that polypronuclear (PPN) embryos positively contribute to the development of the neighboring embryos through secretion of ILIAP, ITI-H4, and keratin. Existence of more than one G4 embryo had a negative effect on the other embryos (p < 0.05). Moreover, G4 embryos were found to secrete KLKB1 and VTDB, which might harm the neighboring embryos. Thus, our study clarified that when embryos are subjected to group culture in WOW-based TLS, the PPN-derived embryos need not be removed, and it is important to ensure that no more than one G4 embryo is present to avoid negative effects on the neighboring embryos.

Comparison of the rates of preterm birth and low birth weight of vanishing twin and primary pregnancies conceived with assisted reproductive technology.

OBJECTIVES: The purpose of this study was to compare the rate of preterm birth, low birth weight, and foetal growth restriction in assisted reproductive technology (ART) singleton pregnancies diagnosed with vanishing twin (VT) syndrome to those of ART pregnancies that were originally singleton pregnancies. METHODS: In this retrospective study, 177 pregnancies diagnosed with VT syndrome were matched and compared with 218 primary singleton pregnancies. The preterm birth and low birth weight rates of these two groups were evaluated. All pregnancies were conceived through ART and delivered at Peking University First Hospital and Hebei Xingtai Infertility Hospital from 2014 to 2016. RESULTS: The preterm delivery rate (20.90 vs. 8.72%, p<0.05) was significantly higher in the ART singletons with VT syndrome than in the control singleton group. The proportion of low-birth-weight (<2500 g) infants was also higher in the VT group than in the primary singleton group (10.73 vs. 3.67%, p<0.05). In addition, the preterm birth rate of the naturally conceived singletons was significantly lower than that of the ART singletons (6.00 vs. 14.18%, p<0.05). CONCLUSIONS: ART singleton pregnancies with VT syndrome have higher rates of preterm birth and low-birth-weight new-borns than ART pregnancies that were originally singleton pregnancies.

Pentachloronitrobenzene alters progesterone production and primordial follicle recruitment in cultured granulosa cells and rat ovary†.

Pentachloronitrobenzene (PCNB) is an organochlorine fungicide widely used for crop production and has become an environmental concern. Little is known about the effect of PCNB on ovarian steroidogenesis and follicular development. We found that PCNB stimulated Star expression and progesterone production in cultured rat granulosa cells in a dose-dependent manner. PCNB activated mitogen-activated protein kinase (MAPK3/1) extracellulat regulated kinase (ERK1/2), thus inhibition of either protein kinase A (PKA) or MAPK3/1 signaling pathway significantly attenuated progesterone biosynthesis caused by PCNB, suggesting that PCNB induced progesterone production by activating the cyclic adenosine monophosphate (cAMP/PKA) and MAPK3/1 signaling pathways. Further investigation demonstrated that PCNB induced Star expression and altered MAPK3/1 signaling in ovary tissues of immature SD rats treated with PCNB at the dose of 100, 200, or 300 mg/kg by daily gavage for 7 days, while serum progesterone level was dose-dependently decreased. We demonstrated that PCNB exposure accelerated the recruitment of primordial follicles into the growing follicle pool in ovary tissues, accompanied by increased levels of anti-Mullerian hormone (AMH) in both ovary tissues and serum. Taken together, our data demonstrate for the first time that PCNB stimulated Star expression, altered MAPK3/1 signaling and progesterone production in vivo and in vitro, and accelerated follicular development with a concomitant increase in AMH in ovary tissues and serum. Our findings provide novel insight into the toxicity of PCNB to animal ovary function.

Discordance between antral follicle counts and anti-Müllerian hormone levels in women undergoing in vitro fertilization.

BACKGROUND: In general, anti-Müllerian hormone (AMH) is positively associated with antral follicle count (AFC). However, there is often discordance between the AMH level and AFC in clinical practice. In cases of discordance, which indicator should be chosen to predict ovarian response and subsequently develop an ovulation induction protocol? The objective of this study was to investigate which indicator was more accurate in predicting ovarian response and pregnancy outcomes when the AMH level and AFC were discordant. METHODS: A total of 1121 infertile women undergoing IVF/ICSI were recruited in this study. During the study period, patients were subjected to individualized controlled ovarian hyperstimulation (COH) protocols according to specific characteristics. The AMH levels and AFCs were measured on days 2-3 of the menstrual cycle. Serum samples were obtained to determine AMH levels. Transvaginal ultrasound was performed to determine the AFC. All patients were divided into four groups: Group A had AFCs and AMH levels in the normal range; Group B had normal AFCs and low AMH levels; Group C had low AFCs and normal AMH levels; and Group D had low AFCs and AMH levels. RESULTS: Two hundred three women (18.11%) showed discordant AFCs and AMH levels. In the two groups with discordant AFCs and AMH levels, namely, Group B and Group C, the oocyte yield, good-quality embryo rate and clinical pregnancy rate were significantly higher in Group B than in Group C. The incidence of poor ovarian response (POR) was significantly lower in Group B than in Group C. According to the stratified analysis of age, for the three categories above the age of 30, oocyte yield was higher in Group B than in Group C. In all age categories, the clinical pregnancy rate was higher in Group B than in Group C. CONCLUSIONS: Our study demonstrated that approximately one in five patients in clinical practice showed discordance between AFCs and AMH levels. In view of the AFC being better than AMH for predicting POR, the AFC should be the preferred indicator for predicting ovarian response to subsequently develop an optimal individualized COH protocol.

Comparison of Connexin43 in Cumulus Cells Between Poor and Non-Poor Responders Undergoing in Vitro Fertilization.

OBJECTIVE: To detect expression of connexin43 in cumulus cells and compare the differences between poor and non-poor ovarian responders undergoing in vitro fertilization (IVF), and to determinate whether the expression of connexin43 correlates with pregnancy outcome. STUDY DESIGN: A total of 152 infertile women undergoing IVF were included in this study. Patients were divided into 2 groups: poor ovarian responders and non-poor ovarian responders. Cumulus cells were collected from oocytes and were analyzed by Western blot for connexin43. Meanwhile, clinical data were obtained. RESULTS: The incidence of poor ovarian response was 18.42% (28/152). Connexin43 was detected in the cumulus cells of all the patients. The relative quantity of connexin43 in the poor responder group (1.11 [0.34, 2.11]) was much lower than that of the non-poor responder group (2.68 [1.41, 4.96]). There was significant difference in connexin43 between pregnant women and non-pregnant women. When adjusting for ovarian response, it yielded similar results. CONCLUSION: Expression of gap junction protein correlates with quantity of oocytes retrieved and might be related to pregnancy outcome. This study suggests that lack of connexin43 expression may contribute to poor ovarian response.

[Testicular histology does not affect the clinical outcomes of ICSI in men with non-obstructive azoospermia].

OBJECTIVE: To investigate whether testicular histology influences the clinical outcomes of intracytoplasmic sperm injection (ICSI) in men with non-obstructive azoospermia (NOA). METHODS: We retrospectively analyzed the clinical data about 73 cases of NOA undergoing ICSI, including 105 ICSI cycles and 79 embryo transfer cycles. The infertility of the patients was attributed to male factors only or both male and female tube factors and the females' age was ≤38 years. Based on testicular histology, we divided the ICSI cycles into three groups: hypospermatogenesis (HS, n = 72), maturation arrest (MA, n = 21) and Sertoli cells only (SCO, n = 12). We recorded and analyzed the age of both the males and females, infertility duration, base follicle-stimulating hormone (FSH) level, dose and days of gonadotropin (Gn) administration, estradiol (E2) and progesterone (P) levels on the day of human chorionic gonadotropin (hCG) administration, endometrial thickness, number of metaphase II (MII) oocytes, and rates of fertilization, transferrable embryos, high-quality embryos, clinical pregnancy, and abortion. RESULTS: The rates of fertilization, failed fertilization, transferrable embryos, and high-quality embryos, and the average number of transferred embryos were 67.03% (553/825), 9.52% (10/105), 85.66% (472/551), 35.03% (193/551), and 2.10, respectively, resulting in 44 pregnancies (55.70%) and 42 live births (53.16%), with no birth defects. No statistically significant differences were observed among the HS, MA and SCO groups in the mean age of the men and women, infertility duration, base FSH level, Gn dose, Gn days, E2 and P levels on the hCG day, endometrial thickness, or number of MII oocytes, nor in the rates of fertilization (68.51% vs 64.39% vs 61.45%), transferrable embryos (85.05% vs 90.48% vs 83.05%), or high-quality embryos (33.09% vs 41.67% vs 38.98%). The rates of clinical pregnancy and embryo implantation were higher in the HS (60.00% and 37.61%) and SCO (62.50% and 50.00%) than in the MA group (37.50% and 21.21%), but with no statistically significant differences (P >0.05). CONCLUSIONS: Once testicular sperm is retrieved, desirable clinical outcomes can be achieved in ICSI for NOA patients, which is not affected by testicular histopathology.

Effect of testosterone on the Connexin37 of sexual mature mouse cumulus oocyte complex.

BACKGROUND: Recent researches demonstrate that pre-treatment with androgen could increase retrieved oocytes number and clinical pregnancy rate in poor ovarian response (POR) patients. In view of gap junction intercellular communication (GJIC) is important for follicular growth, and androgen plays an important role in improving prognosis of POR patients, we speculate that androgen can increase the expression of connexin in follicle cells, and improve ovarian microenvironment, thus can promote ovarian response. The objective of the research is to study the effect of testosterone on connexin37 (Cx37) expression so as to provide theoretical basis for adding testosterone in treatment of POR. METHODS: Cumulus-oocyte-cells (COCs) were collected from ICR mice ovaries, and were cultured in vitro for 48 h and then treated with testosterone (T) at various concentration. To assess whether the effect of androgen on Cx37 expression is mediated through androgen receptor (AR) pathway, COCs were cultured in vitro with Flutamide (androgen receptor antagonist). The expression of Cx37 was determined by western blot. RESULTS: The expression of Cx37 in COCs which were treated with testosterone was higher than that of control group. There were significant differences (P < 0.001;<0.001;<0.001;<0.001). Cx37 increased with the elevated testosterone concentrations. Cx37 was lower in androgen receptor antagonist group (2.57 ± 0.12) than the corresponding testosterone concentrations group (4.42 ± 0.28). There were significant differences between two groups (P < 0.001). CONCLUSIONS: There was close relationship between gap junction protein and ovarian response, which suggested that androgen could promote ovarian response by increasing the expression of Cx37 in follicle. Androgen plays an important role in ovarian response through the AR pathway and non-AR pathway.

[Testicular sperm cryopreservation for male fertility preservation].

OBJECTIVE: To investigate the effectiveness of testicular sperm cryopreservation in male fertility preservation by evaluating the clinical outcome of ICSI cycles with frozen-thawed testicular sperm for azoospermia patients. METHODS: We retrospectively analyzed 96 samples of cryopreserved testicular sperm obtained by testicular biopsy, vasovasostomy (V-V), vasoepididymostomy (V-E) , of which 55 were subjected to 60 ICSI cycles with frozen-thawed testicular sperm. We evaluated the rates of sperm recovery, fertilization, cleavage, transferable and good-quality embryos, clinical pregnancy, pregnancy outcome, and health of the newborns. RESULTS: All the frozen testicular sperm samples were recovered successfully. The rates of fertilization, 2PN fertilization, cleavage, available embryos and good-quality embryos were 77.6, 69.4, 99.4, 84.5 and 40.8%, respectively. There were transferable embryos in all cycles. Fresh embryos were transferred in 52 of the 60 cycles, with the clinical pregnancy rate of 57.7% (30/52), including 19 singletons and 11 twins, and the rates of implantation and miscarriage were 38.7% (41/106) and 3.33% (1/30). Up to the present time, there have been 20 healthy newborns, including 12 boys and 8 girls, and another 13 ongoing pregnancies. No birth defects have been found so far. CONCLUSION: Desirable clinical outcomes can be obtained from ICSI cycles with frozen-thawed testicular sperm, and testicular sperm cryopreservation is an effective method of fertility preservation for azoospermia males.

Translocation of classical PKC and cortical granule exocytosis of human oocyte in germinal vesicle and metaphase II stage.

AIM: Protein kinase C (PKC) is as a family of serine/threonine kinases that can be activated by Ca2+, phospholipid and diacylglycerol. PKC plays an important role in oocyte maturation and activation. This study was undertaken to investigate classical PKC (cPKC) in human oocyte maturation and activation. METHODS: Germinal vesicle (GV) and metaphase II (MII) stage oocytes were collected from healthy women. The expression and distribution of cPKC were investigated by immunoflourescence. MII oocytes were treated with PKC activator or inhibitor and imaged using a laser confocal scanning microscope (LCSM). RESULTS: In GV oocytes, PKCalpha, beta1 and gamma were localized to the germinal vesicles, with a weak expression in ooplasm. In MII oocytes, PKCalpha, beta1 and gamma were distributed evenly in ooplasm. After treatment with PKC activator, phorbol 12-myristate 13-acetate (PMA), cPKC translocated to the periphery of oocyte, and cortical granules (CG) exocytosis was found. When the oocytes were treated with PKC inhibitor, staurosporine, no translocation of cPKC and CG exocytosis were found. CONCLUSION: PKCalpha, beta1 and gamma exist in human oocytes and activation of these subunits could induce CG exocytosis in MII stage.